Comparison of human red cell lysis by hypochlorous and hypobromous acids: insights into the mechanism of lysis.
Human red blood cells are lysed by the neutrophil-derived oxidant hypochlorous acid (HOCl), although the mechanism of lysis is unknown. Hypobromous acid (HOBr), a similarly reactive oxidant, lysed red cells approx. 10-fold faster than HOCl. Therefore we compared the effects of these oxidants on thiols, membrane lipids and proteins to determine which reactions are associated with lysis. There was no difference in the loss of reduced glutathione or membrane thiols with either oxidant, but HOBr reacted more readily with membrane lipids and proteins. Bromohydrin derivatives of phospholipids and cholesterol were seen at approx. one-tenth the level of oxidant than chlorohydrins were. However, these products were detected only with high concentrations of HOCl or HOBr, which caused instant haemolysis. Membrane protein modification occurred at much lower doses of oxidant and was more closely correlated with lysis. SDS/PAGE analysis showed that band 3, the anion transport protein, was lost at the lowest dose of HOBr and at the higher concentrations of HOCl. Labelling the red cells with eosin 5-maleimide, a fluorescent label for band 3, suggested possible clustering of this protein in oxidant-exposed cells. There was also irreversible cross-linking of all the major membrane proteins; this reaction occurred more readily with HOBr. The results indicate that membrane protein modification is the reaction responsible for HOCl-mediated lysis. These effects, and particularly cross-link formation, might result in clustering of band 3 and other membrane and cytoskeletal proteins to form haemolytic pores.
Vissers
M C
MC
Free Radical Research Group, Department of Pathology, Christchurch School of Medicine, P.O. Box 4345, Christchurch, New Zealand.
Carr
A C
AC
Chapman
A L
AL
D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry; D001959:Bromates / Q000494:pharmacology*; D003432:Cross-Linking Reagents; D004910:Erythrocyte Membrane / Q000737:chemistry*; D006461:Hemolysis / Q000187:drug effects*; D006801:Humans; D006997:Hypochlorous Acid / Q000494:pharmacology*; D008563:Membrane Lipids / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D016877:Oxidants / Q000494:pharmacology*; D010084:Oxidation-Reduction; D013431:Sulfates / Q000378:metabolism
D001457:Anion Exchange Protein 1, Erythrocyte; D001959:Bromates; D003432:Cross-Linking Reagents; D008563:Membrane Lipids; D008565:Membrane Proteins; D016877:Oxidants; D013431:Sulfates; D006997:Hypochlorous Acid; C027664:hypobromous acid